High performance liquid chromatography is effected generally by percolating a number of component solute molecules in a flowing stream of a liquid through a packed bed of particles, known as the stationary phase, which efficiently separates the various types of molecules from one another.
Such separations, particularly in the preparative area, have significant limitations typically occasioned by the batch nature of the current processes. Typically, a chromatographic column is first equilibrated by flowing a liquid through the column. The latter is then charged or loaded with a fluid mixture containing the solute or solutes sought to be separated, and one or more eluant liquids or an eluant gradient are run sequentially through the column to release bound solute selectively. The eluted solutes, usually in relatively minute quantities, are thus temporally separated at the output of the column and the process is repeated cyclically. It is highly desirable to verify the intended changes in solution composition that actually arrive at the inlet of the chromatographic column in the intended manner. It may also be desirable to monitor the time-varying output of the column which provides an indication of band-spreading due to mixing of adjacent volumes of different solvents in the input conduits and the column itself, and malfunctions in the eluant gradient generating devices or other malfunctions which may cause undesirable behavior of the system. In chromatographic systems typical of the prior art, the electrical input used to control the gradient generator also generate the display of the gradient profile, and does not provide any measurement of the actual fluid output of the gradient generator. Consequently, malfunctions (even as common and trivial as running out of solvent) that create gradient conditions other than those intended, will only be noted when the chromatographic separation fails to yield the desired result, i.e. when it is too late to resolve.
Conventional or standard detectors are designed generally to be installed downstream from the chromatographic column where the pressure level is the lowest in the system and where the desired solute has been diluted as a result of the separation process. The material or solute, as it is detected at the outlet of the column, is generally in a much larger volume of solvent than when it is introduced at the input to the column. The greater volume of the peak to be detected at the column outlet means that band-spreading in the detector can be greater without any major effect on the overall separation, and because the more dilute conditions require a more sensitive detector, usually a larger detector cell is provided which tends to produce even more band-spreading. Since the pressure requirements imposed on detectors placed at the column output are very modest, the detector designs are slanted toward high sensitivity and it is doubtful that currently used detectors are capable of sustaining the pressure levels commonly present at the inlet to HPLC columns. Even if such detectors were strengthened to meet such pressure demands, because they tend to introduce excessive band-spreading, they would not adequately serve as detectors for the column inlet.
The foregoing considerations are, a fortiori, important in the context of certain novel methods and apparatus for performing HPLC at the very high inlet pressures required to insure that flow through the chromatographic column occurs at a reduced velocities of greater than about 5,000, a highly efficient chromatography system described more fully in U.S. Pat. Nos. 5,772,874, 5,795,469 and 5,919,368, the same being incorporated in their entirety herein by reference.